Project Summary/ Abstract Tuberculosis (TB) is a common and potentially severe illness in HIV-infected children. Diagnosis of pediatric TB in the context of HIV is challenging due to non specific clinical and radiological changes, anergy to the tuberculin skin test (TST) and the difficulty of microbiological confirmation. Nevertheless, establishing a timely diagnosis of TB in HIV-infected children is crucial to prevent HIV progression or development of severe tuberculous disease. The aim of this study is to improve the diagnosis of TB in HIV-infected children. This will be done through 2 approaches: A microbiological and molecular approach (to detect the TB organism, TB-DNA or TB-antigen) will include an evaluation of: (i) two novel DNA-amplification methods (loop mediated isothermal amplification [LAMP], and Xpert MTB [based on real-time PCR]) which provide significant advances over current technology by minimizing operator dependence, improving DNA extraction and simplifying amplicon detection; and an antigen detection method (urinary lipoarabinomannan) for rapid diagnosis (within 1 day) of childhood TB disease; (ii) the use of novel specimens (induced sputum and nasopharyngeal aspirates) and new culture techniques (microscopic observation drug susceptibility [MODS] assay) compared with conventional (mycobacterial growth indicator [MGIT] culture) to improve the yield and speed of microscopy and culture-based diagnosis of childhood TB. An immunological approach (to detect the child's specific immune response to TB) will include an evaluation of: (i) the incremental value of adding TB-specific ELISpot instead of or with TST to clinical diagnostic algorithms for TB disease in HIV-infected children; and (ii) the use of ELISpot on cells from a site-specific specimen (e.g. pleural or cerebrospinal fluid) compared with ELISpot using peripheral blood for the diagnosis of extrapulmonary TB. We propose to investigate these approaches in a prospective study of 700 consecutive children (500 HIV- infected) hospitalised with suspected pulmonary or extrapulmonary TB in Cape Town, South Africa. Children will undergo routine and study specific investigations and will be followed for their response to therapy to define a `gold-standard' reference group of children with culture-confirmed TB and a second group in which TB has been definitively excluded. These groups will be used to determine the sensitivity and specificity of the novel tests under evaluation. The results of this study should lead to an enhanced clinical diagnostic algorithm and improved, more rapid microbiological tests for the diagnosis of TB in HIV-infected children. Project Narrative TB is a common and severe illness in HIV-infected children. Diagnosis in HIV-infected children is difficult; timely diagnosis is essential to prevent HIV progression or development of severe TB. The results of this study will lead to improved clinical methods of diagnosis and provide better, more rapid microbiological methods for confirmation of TB and for TB drug resistance testing in HIV-infected children. Early case detection and implementation of effective therapy will reduce morbidity and mortality in HIV-infected children, reduce the potential for TB transmission by identifying source adult cases and improve TB control.